The production of industrial enzymes by culturing microorganisms, such as bacteria, fungi and yeast, in aqueous nutrient media is well-known. Depending on the nature of the particular microorganism, the enzyme(s) produced may be extracellular, intracellular, or a mixture thereof. When the enzyme(s) produced are extracellular, they are generally obtained by first separating the enzyme-containing supernatant from the microorganism cells and then recovering the enzyme(s) from the supernatant by well-known methods, such as precipitation, ultrafiltration and evaporation. When the enzyme(s) produced are intracellular, they must first be released from the cells. This can be accomplished chemically and/or mechanically. Once the enzyme(s) are placed into a solution, they can also be recovered by the above well-known methods.
It is known that extracellular enzymes have compositions and properties which are different from intracellular enzymes. Extracellular enzymes, for example, generally have significantly lower molecular weights than intracellular enzymes. The solution containing intracellular enzymes released from the cells will also contain significant quantities of other intracellular material that are not present in a whole fermentation beer containing extracellular enzymes. These differences can cause different procedures to be employed for recovery and purification thereof even when they are both in aqueous solution. A recovery procedure suitable for intracellular enzymes is thus not obviously useful for extracellular enzymes.
In order to improve the efficiency and convenience of enzyme production and recovery, it is known that enzymes can be produced by a microorganism fermentation in a two-phase nutrient medium containing a mixture of polyethylene glycol and dextran. At the completion of the fermentation, the extracellular enzyme is concentrated in the upper polyethylene glycol phase while the cells and other fermentation products are concentrated in the lower dextran phase. This is described in Enzyme and Microb. Technol. Vol. 7, 333-338 (1985). The partition coefficient for alpha-amylase in that system had a maximum of 4, for example.
A variation of the above procedure is described in U.S. Pat. No. 4,508,825 wherein the extracellular enzyme-containing supernatant is separated from the cells, and the cell-free supernatant is mixed with polyethylene glycol and a cationic epihalohydrin/polyamine copolymer or dextran polymer to form two phases. This technique can be used to separate extracellular protease and amylase wherein the protease is concentrated in the polyethylene glycol phase and the amylase is concentrated in the cationic copolymer or dextran phase.
Two-phase enzyme recovery procedures have also been used with intracellular enzymes. U.S. Pat. No. 4,144,130 describes the use of (1) a mixture of a high molecular weight unsubstituted or substituted polyalcohol, polyether, polyester, polyvinylpyrrolidone or polysaccharide and an inorganic salt, or (2) a mixture of at least two of the above high molecular weight polymers to recover intracellular enzymes from an aqueous solution into which they have been released from the cells. When a mixture of polyethylene glycol and an inorganic salt, for example, is used, the desired intracellular enzyme goes into the top polyethylene glycol layer while the cell debris and other fermentation products go into the lower salt-containing layer. The partition coefficient for various enzymes recovered in the glycol layer was about 0.3 when a normal cell mass was treated. The partition coefficient was increased to only about 3 when frozen cells were mixed with water and disintegrated to release their enzymes.
The addition of polyethylene glycol to assist inorganic salts in the precipitation of enzymes from cell-free supernatant is disclosed in U.S. Pat. No. 4,016,039.
There has been no suggestion in the prior art that polyethylene glycol and inorganic salts could be used in a two-phase process to recover extracellular enzymes from whole fermentation beer with partition coefficients of at least 50.